Cellular Seeding and Co-Culture of a Three Dimensional Fibroblast Construct

ABSTRACT

The present invention provides methods for cellular seeding onto three-dimensional fibroblast constructs, three-dimensional fibroblast constructs seeded with muscle cells, and uses therefore.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/260,610, filed Feb. 7, 2012, which is a US national stage application of PCT Application No. PCT/US10/30579 filed Apr. 9, 2010, which claims the benefit of U.S. Provisional Patent Application Ser. No. 61/212,280 filed Apr. 9, 2009, incorporated by reference herein in its entirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under Merit Review Grant #0128 awarded by Veterans Administration. The U.S. government has certain rights in the invention.

SEQUENCE LISTING STATEMENT

The sequence listing is filed in this application in electronic format only and is incorporated by reference herein. The sequence listing text file “10-214-PCTseqlist_ST25.txt” was created on Apr. 9, 2010, and is 32,609 byte in size.

BACKGROUND

New treatments are needed for patients with chronic heart failure (CHF), the No. 1 hospital discharge diagnosis in patients over the age of 65 years of age in this country, as well as related ischemic and non-ischemic cardiac disorders. The prevalence of heart failure is over 5 million; the incidence is 550,000 patients per year. Heart failure results in more deaths than cancer, accidents, and strokes combined, costing more than $23 billion annually. Once a patient becomes symptomatic with NY Class III or IV heart failure, their mortality approaches 50% in two years without a heart transplant. The newest approach to treat CHF is to inject stem cells and/or progenitor cells directly into the heart using a number of different cell types. However, the results from recent clinical trials using such injection strategies are generally disappointing.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides constructs comprising muscle cells adhered to a 3DFC, wherein the construct is capable of spontaneous synchronized contractions across the 3DFC; and wherein the muscle cells are seeded on the construct at a density of between 0.5×10⁶ cells/cm² and 5×10⁶ cells/cm² and/or the muscle cells are present in a ratio of between about 1:10 and about 10:1 with fibroblasts on the 3DFC. In one embodiment, the muscle cells comprise cardiomyocytes or cardiac stem cells.

In a second aspect, the present invention provides methods for treating a disorder characterized by a lack of functioning cardiomyocytes, comprising contacting the heart of a subject suffering from such a disorder with an amount effective to treat the disorder of a construct of any embodiment of the first aspect of the invention. In various embodiments, the disorder is selected from the group consisting of chronic heart failure (CHF), ischemia without heart failure, cardiomyopathy (such as dilated cardiomyopathy (DCM)), cardiac arrest, congestive heart failure, stable angina, unstable angina, myocardial infarction, coronary artery disease, valvular heart disease, ischemic heart disease, reduced ejection fraction, reduced myocardial perfusion, maladaptive cardiac remodeling (such as left ventricle remodeling), reduced left ventricle function, left heart failure, right heart failure, backward heart failure (increased venous back pressure), forward heart failure (failure to supply adequate arterial perfusion), systolic dysfunction, diastolic dysfunction, systemic vascular resistance, low-output heart failure, high-output heart failure, dyspnea on exertion, dyspnea at rest, orthopnea, tachypnea, paroxysmal nocturnal dyspnea, dizziness, confusion, cool extremities at rest, exercise intolerance, easy fatigueability, peripheral edema, nocturia, ascites, hepatomegaly, pulmonary edema, cyanosis, laterally displaced apex beat, gallop rhythm, heart murmurs, parasternal heave, and pleural effusion.

In a third aspect, the present invention provides methods for seeding a three dimensional fibroblast construct (3DFC) with cells, comprising:

(a) contacting a cultured 3DFC with a suspension of cells to be seeded onto the 3DFC;

(b) subjecting the cells within the suspension to a force that causes said cells to contact the 3DFC; and

(c) culturing the cells under conditions suitable for the cells to adhere to the 3DFC.

In a fourth aspect, the present invention provides methods for seeding a three dimensional fibroblast construct (3DFC) with cells, comprising:

(a) contacting a cultured 3DFC with a cell sheet to be seeded onto the 3DFC; and

(b) culturing the cell sheet under conditions suitable for the cell sheet to adhere to the 3DFC.

In a fifth aspect, the present invention provides constructs of any embodiment of the first aspect of the invention made using the methods of any embodiment of the third or fourth aspects of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Endothelial progenitor cells tagged with CFDA-se co-cultured on 3DFC at A) 10 min and B) 24 hrs after seeding. Positively stained CFDA-se tagged cells fluoresce green demonstrating additional cells seeded on the 3DFC survive and proliferate. The biodegradable vicryl mesh can be seen in the background.

FIG. 2: Endothelial progenitor cells in a culture plate following isolation of an intact cell sheet. Cells maintain all cell-to-cell adhesion molecules and can be seeded and co-cultured onto the 3DFC.

FIG. 3: Endothelial progenitor cell sheet after H&E stain. Dark purple represents nuclei while the lighter pink indicates cytoplasm. Image shows (A) the full cell sheet (−8 mm diameter) and (B&C) higher magnification.

FIG. 4: FIG. 4: 40× time elapsed images of control (A), NCM-3DFC (B), and NCM-3DFC+halothane (C) treated patches, cultured for 6 days and continuously injected with NBD. (A) Injection of a single fibroblast, note lack of dye transfer. (B) Injection of single neonatal cardiomyocyte on NCM-3DFC spreads to numerous neighboring cells. (C) Injection of a single neonatal cardiomyocyte on NCM-3DFC treated with halothane. Note the disruption of dye transfer due to blockage of connexins by halothane. Cells regain ability to use gap junctions 15 to 20 minutes after halothane treatment.

FIG. 5: Patches seeded with cardiomyocytes improved EF 25% in treated vs. sham rats after 3 wks. Data are mean±SE/. NCM-3DFC=Neonatal Cardiomyocyte 3DFC; SO=Sham Operated. SO, n=21; UN, n=12; 3DFC, n=9; NCM-3DFC, n=9. * P<0.05 vs SO; @ P<0.05 vs UN; # P<0.05 vs 3DFC.

FIG. 6: Patches seeded with cardiomyocytes improved cardiac index (ml/(min X grams)) 55% in treated vs. sham rats after 3 wks. Data are mean±SE. NCM-3DFC=Neonatal Cardiomyocyte 3DFC; SO=Sham Operated. SO, n=21; UN, n=12; 3DFC, n=9; NCM-3DFC, n=9. * P<0.05 vs SO; @ P<0.05 vs UN; # P<0.05 vs 3DFC.

FIG. 7: Patches seeded with cardiomyocytes improved end diastolic pressure (mmHg) in treated vs. sham rats after 3 wks. Data are mean±SE. NCM-3DFC=Neonatal Cardiomyocyte 3DFC; SO=Sham Operated. SO, n=19; UN (untreated), n=12; 3DFC, n=9; NCM-3DFC, n=9. * P<0.05 vs SO; @ P<0.05 vs UN; # P<0.05 vs 3DFC.

FIG. 8: Hematoxylin and eosin stained 3DFC (A) and cardiomyocyte seeded 3DFC (B & C). 10× magnification of 3DFC (A) orientated horizontally. Vicryl bundles (in purple) and fibroblasts nuclei blue/purple dots. 5× magnification of cardiomyocyte seeded 3DFC (B) orientated horizontally. 10× magnification of cardiomyocyte seeded 3DFC (C) orientated horizontally. The large cellular bundles (B & C) consist of cardiomyocytes situated between vicryl fibers.

FIG. 9: 10× magnification H&E of 3DFC orientated horizontally. Vicryl bundles (in purple) and fibroblasts nuclei blue/purple dots.

FIG. 10: 5× magnification H&E of cardiomyocyte seeded 3DFC orientated horizontally. The large cellular bundles consist of cardiomyocytes situated between vicryl fibers.

FIG. 11: 10× magnification H&E of cardiomyocyte seeded 3DFC orientated horizontally. The large cellular bundles consist of cardiomyocytes situated between vicryl fibers.

DETAILED DESCRIPTION OF THE INVENTION

All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), “Guide to Protein Purification” in Methods in Enzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.).

As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. “And” as used herein is interchangeably used with “or” unless expressly stated otherwise.

All embodiments disclosed herein can be combined with other embodiments unless the context clearly dictates otherwise.

In a first aspect, the present invention provides methods for seeding a three dimensional fibroblast construct (3DFC) with cells, comprising:

(a) contacting a cultured 3DFC with a suspension of cells to be seeded onto the 3DFC;

(b) subjecting the cells within the suspension to a force that causes the cells to contact the 3DFC; and

(c) culturing the cells under conditions suitable for the cells to adhere to the 3DFC.

The inventors have discovered that cells to be adhered to the 3DFC do not adequately settle out of solution onto the patch and adhere to the 3DFC through cell-surface adhesion molecules. Early attempts at seeding the 3DFC evaluated the placement of cells suspended in culture media with the notion that standard gravitational force would “settle” the desired cells on the 3DFC below. Results found, poor retention of cells on the 3DFC with a majority of cells passing to the culture plate below. The inventors thus developed the methods of this first aspect of the invention to force the cells to adhere to the surface of the 3DFC. Such seeded 3DFC can be used, for example, in cell-based therapies, as described in detail below.

As used herein, a “three dimensional fibroblast construct” is a construct comprising fibroblasts grown on a three-dimensional substrate comprising a biocompatible, non-living material formed into a three-dimensional structure having interstitial spaces bridged by the cells in the construct. It will be understood that the 3DFC may contain cell types in addition to fibroblasts as appropriate for a given purpose. For example, the 3DFC may also comprise other stromal cells, including but not limited to endothelial cells. See, for example, published US patent application US2009/0269316 and U.S. Pat. No. 4,963,489, both incorporated by reference herein in their entirety.

The fibroblasts and other cells may be fetal or adult in origin, and may be derived from convenient sources such as skin, cardiac muscle, smooth muscle, skeletal muscle, liver, pancreas, brain, adipose tissue (fat) etc. Such tissues and or organs can be obtained by appropriate biopsy or upon autopsy. In alternative embodiments for all aspects of the invention, the fibroblasts and other cells are human cells. In an alternative embodiment for all aspects of the invention, the 3DFC is a matrix-embedded human dermal construct of newborn dermal fibroblasts cultured in vitro onto a bioabsorbable mesh to produce living, metabolically active tissue. The fibroblasts proliferate across the mesh and secrete a large variety of growth factors and cytokines, including human dermal collagen, fibronectin, and glycosaminoglycans (GAGs), embedding themselves in a self-produced dermal matrix. In culture the fibroblasts produce angiogenic growth factors: VEGF (vascular endothelial growth factor), HGF (hepatocyte growth factor), bFGF (basic fibroblast growth factor), and angiopoietin-1 (See, for example, J. Anat. (2006) 209, pp 527-532)

The cells to be seeded onto the 3DFC may be of any desired type, including but not limited to muscle cells (skeletal muscle cells, smooth muscle cells, cardiac muscle cells such as cardiomyocyte) or progenitors thereof, endothelial progenitor cells, bone marrow cells, bone marrow cells, mesenchymal stem cells, umbilical cord blood cells or combinations thereof. Such cells can be isolated using standard techniques in the art, or may be obtained from commercial sources.

In an alternative embodiment of all aspects of the invention, the seeded cells comprise cardiomyocytes and/or progenitors thereof such as cardiac stem cells. There are a limited number of intrinsic cardiac stem cells in the mature adult heart that are self-renewing, clonogenic, and multipotent, such that they differentiate into cardiomyocytes and, to a lesser extent, into smooth muscle and endothelial cells. Cardiac stem cells can be isolated and expanded in culture indefinitely. In one embodiment, the cardiac stem cells are characterized by cell surface markers: Lin−, c-Kit+, CD45−, CD34−.

The cells to be seeded may be recombinant cells capable of expressing a gene product of interest for a given purpose. In one alternative embodiment as described in more detail below, cardiomyocytes engineered to express one or more of thymosin beta-4 (TB4), akt murine thyoma viral oncogene homolog (AKT1; SEQ ID NO:1, 3, or 5 for amino acid sequence), stroma cell-derived factor-1 alpha (SDF-1; SEQ ID NO:7 for amino acid sequence), and hepatocyte growth factor (HGF; SEQ ID NO:9 for amino acid sequence) are seeded onto the 3DFC. Techniques for engineering cells to express a heterologus gene product are well known in the art, and utilize cell transfection by recombinant expression vector that operatively link a nucleic acid coding region or gene to any promoter capable of effecting expression of the gene product. Thus, in various embodiments, the cells comprise a recombinant expression vector encoding a nucleic acid sequence that encodes the polypeptide sequence one or more of SEQ ID NO:1, 3, 5, 7, 9, and 11, In various further embodiments, the nucleic acid sequence is selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, and 10. The promoter sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF, etc.) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). See, for example, Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.).

Contacting a cultured 3DFC with a suspension of cells to be seeded onto the 3DFC can be done under any suitable conditions to facilitate application of the force that causes the cells to contact the 3DFC, including but not limited to the conditions described below. In one alternative embodiment, the 3DFC is placed in 0.1 to 5 ml of media (preferably 0.25 ml-2 ml, and more preferable 0.5 ml-1.0 ml of media), and cells are introduced in suspension, such that the volume of cell suspension is approximately double the volume of media in which the 3DFC is placed. In one alternative embodiment that can be combined with any other embodiments herein, the contacting occurs at approximately 37° C. In a further alternative embodiment that can be combined with any other embodiments herein, the cell suspension has a concentration of at least 3×10⁶ cells/ml where the cells to be seeded are contractile cells (such as cardiomyocytes or progrenitors thereof). In further alternative embodiments that can be combined with any other embodiments herein, the cell suspension has a concentration of at least 4×10⁶ cells/ml and 5×10⁶ cells/ml.

In one embodiment, each 3DFC to be seeded is placed in a well so as to cover the base of the well and lay flat. The inventors have discovered that if the 3DFC does not cover the base of the well (when seeding 3DFCs placed in wells), a decreased retention of cells occurs, and results in an unequal distribution of cells across the patch due to cell bunching and clumping.

Subjecting the cells within the suspension to a force that causes said cells to contact the 3DFC may comprise the use of any suitable force, including but not limited to a centrifugal force and an electrical force generated by an electric field, or combinations of such forces. In an alternative embodiment, a centrifugal force is used. The centrifugal force to be applied depends on a variety of factors, such as the cell type to be seeded onto the 3DFC. In one alternative embodiment that can be combined with any other embodiments herein, the construct is centrifuged at between 1200 rpms and 1600 rpms for between 2 and 10 minutes. In an alternative embodiment, all 3DFC constructs to be seeded are placed in a horizontal arrangement in wells (as opposed to vertical), so that each well is spun at the same radius.

In one alternative embodiment, the force may be applied within 0-300 seconds after contacting of the cell suspension with the 3DFC in appropriate culture medium.

It will be understood by those of skill in the art that it is not a requirement that all cells in the suspension contact the 3DFC as a result of the force application, as the cells can preferably be present in the suspension in an amount that saturates all available locations for seeding onto the 3DFC. In one alternative embodiment, the seeded cells contact each other, such that multiple cell layers are provided on top of the 3DFC. In embodiments where cardiomyocytes or precursors thereof are used, it is preferred that the seeded cells reside in the “valleys” between fibers on the 3DFC (see below for description of 3DFC structure). Exemplary images of such cardiomyocyte-seeded 3DFCs are shown in FIGS. 8-11.

Culturing the cells under conditions suitable for the cells to adhere to the 3DFC may comprise the use of any culture media and conditions suitable for a given purpose, such as those in the examples that follow. Any useful media may be used, including but not limited to DMEM-LG supplemented with fetal bovine serum (5-15%; preferably 10%) and other appropriate factors (including but not limited to sodium bicarbonate and antibiotics. It will be understood by those of skill in the art that it is not a requirement that all cells in the suspension adhere to the 3DFC as a result of the force application, as the cells can preferably be present in the suspension in an amount that saturates all available locations for adherence onto the 3DFC. In an alternative embodiment that can be combined with any other embodiment disclosed herein, the cells are adhered to the 3DFC at a cell density ranging between 0.5×10⁶ cells/cm² and 5×10⁶ cells/cm²; more preferably between 1×10⁶ cells/cm² and 4×10⁶ cells/cm²; and most preferably between 1.5×10⁶ cells/cm² and 3×10⁶ cells/cm².

In a further alternative embodiment that can be combined with any of the other embodiments disclosed herein, the culturing further comprises growth of cells adhered to the 3DFC. In this embodiment, such growth can occur under the same or different culture conditions than those used to promote adherence of the suspended cells to the 3DFC. Suitable culture conditions to promote proliferation and/or differentiation of cells adhered to the 3DFC can be determined by those of skill in the art, based on the disclosure herein. In another alternative embodiment that can be combined with any other embodiment herein, the seeded 3DFCs are incubated within 5 minutes of application of force and not disturbed (i.e. the 3DFCs are not removed from culture plate, media changed, etc.). Seeded 3DFCs for in vivo implant are preferably harvested from culture plates ˜18-20 hrs after seeding/incubation.

Exemplary methods for seeding are provided in the examples that follow. The 3DFC may be purchased from commercial sources (ANGINERA™ (Theregen, Inc., California); Advanced BioHealing, Inc (DERMAGRAFT®)), or may be prepared as described, for example, in US2009/0269316 and U.S. Pat. No. 4,963,489. Briefly, fibroblasts and, optionally, other stromal cells as deemed appropriate for a given purpose, are inoculated upon a three-dimensional framework, and grown to develop the 3DFC. The fibroblasts and other stromal cells may be engineered to express gene products of interest, such as thymosin beta 4. See, for example, U.S. Pat. Nos. 5,785,964 and 5,957,972, incorporated by reference herein in their entirety.

The three-dimensional support framework may be of any material and/or shape that: (a) allows cells to attach to it (or can be modified to allow cells to attach to it); and (b) allows cells to grow in more than one layer. A number of different materials may be used to form the framework, including but not limited to: nylon (polyamides), dacron (polyesters), polystyrene, polypropylene, polyacrylates, polyvinyl compounds (e.g., polyvinylchloride; PVC), polycarbonate, polytetrafluorethylene (PTFE; TEFLON), thermanox (TPX), nitrocellulose, cotton, polyglycolic acid (PGA), cat gut sutures, cellulose, gelatin, dextran, etc. Any of these materials may be woven into a mesh to form the three-dimensional framework. Certain materials, such as nylon, polystyrene, etc., are poor substrates for cellular attachment. When these materials are used as the three-dimensional support framework, it is advisable to pre-treat the framework prior to inoculation of fibroblasts and other stromal cells in order to enhance their attachment to the framework. For example, prior to inoculation with fibroblasts and other stromal cells, nylon screens could be treated with 0.1 M acetic acid, and incubated in polylysine, fetal bovine serum, and/or collagen to coat the nylon. Polystyrene could be similarly treated using sulfuric acid.

When the 3DFC is to be implanted directly in vivo, it may be preferable to use biodegradable materials such as PGA, catgut suture material, collagen, polylactic acid, or hyaluronic acid. For example, these materials may be woven into a three-dimensional framework such as a collagen sponge or collagen gel. Where the cultures are to be maintained for long periods of time or cryopreserved, non-degradable materials such as nylon, dacron, polystyrene, polyacrylates, polyvinyls, teflons, cotton, etc. may be preferred. A convenient nylon mesh which could be used in accordance with the invention is a nylon filtration mesh having an average pore size of 140 μm and an average nylon fiber diameter of 90 μm (#3-210/36, Tetko, Inc., N.Y.).

Stromal cells comprising fibroblasts, with or without other cells and elements described below, are inoculated onto the framework. These stromal cells may be derived from tissues or organs, such as skin, heart, blood vessels, skeletal muscle, liver, pancreas, brain etc., which can be obtained by biopsy (where appropriate), from surgically obtained specimens, or upon autopsy. Fetal fibroblasts can be used to form a “generic” three-dimensional stromal tissue that will support the growth of a variety of different cells and/or tissues that come in contact with it. However, a “specific” stromal tissue may be prepared by inoculating the three-dimensional framework with stromal cells derived from the heart and/or from a particular individual who is later to receive the cells and/or tissues grown in culture in accordance with the present invention. Such samples can be obtained, for example, through standard biopsy procedures (such as a myocardial biopsy when stromal cells from the heart are to be obtained) or other surgical procedure.

Stromal cells may be readily isolated by disaggregating an appropriate organ or tissue. This may be readily accomplished using techniques known to those skilled in the art. For example, the tissue or organ can be disaggregated mechanically and/or treated with digestive enzymes and/or chelating agents that weaken the connections between neighboring cells making it possible to disperse the tissue into a suspension of individual cells without appreciable cell breakage. Enzymatic dissociation can be accomplished by mincing the tissue and treating the minced tissue with any of a number of digestive enzymes either alone or in combination. These include, but are not limited to, trypsin, chymotrypsin, collagenase, elastase, and/or hyaluronidase, DNase, pronase, and dispase. Mechanical disruption can also be accomplished by a number of methods including, but not limited to, the use of grinders, blenders, sieves, homogenizers, pressure cells, or insonators to name but a few. For a review of tissue disaggregation techniques, see Freshney, Culture of Animal Cells. A Manual of Basic Technique, 2d Ed., A.R. Liss, Inc., New York, 1987, Ch. 9, pp. 107-126.

Once the tissue has been reduced to a suspension of individual cells, the suspension can be fractionated into subpopulations from which the fibroblasts and/or other stromal cells and/or elements can be obtained. This also may be accomplished using standard techniques for cell separation including, but not limited to, cloning and selection of specific cell types, selective destruction of unwanted cells (negative selection), separation based upon differential cell agglutinability in the mixed population, freeze-thaw procedures, differential adherence properties of the cells in the mixed population, filtration, conventional and zonal centrifugation, centrifugal elutriation (counter-streaming centrifugation), unit gravity separation, countercurrent distribution, electrophoresis and fluorescence-activated cell sorting. For a review of clonal selection and cell separation techniques, see Freshney, Culture of Animal Cells. A Manual of Basic Techniques, 2d Ed., A.R. Liss, Inc., New York, 1987, Ch. 11 and 12, pp. 137-168.

Where the cultured cells are to be used for transplantation or implantation in vivo, it is preferable to obtain the stromal cells from the patient's own tissues.

After inoculation of the stromal cells, the 3DFC should be incubated in an appropriate nutrient medium. Many commercially available media such as RPMI 1640, Fisher's, Iscove's, McCoy's, and the like may be suitable for use. In an alternative embodiment, the 3DFC may be suspended in the medium during the incubation period in order to maximize proliferative activity of the fibroblasts and other stromal cells. In addition, the culture should be “fed” periodically to remove the spent media, depopulate released cells, and add fresh media. During the incubation period, the fibroblasts and other stromal cells will grow linearly along and envelop the filaments of the three-dimensional framework before beginning to grow into the openings of the framework.

The openings of the framework should be of an appropriate size to allow the fibroblasts and other stromal cells to stretch across the openings. Maintaining actively growing stromal cells that stretch across the framework enhances the production of growth factors that are elaborated by the stromal cells, and hence will support long term cultures. For example, if the openings are too small, the stromal cells may rapidly achieve confluence but be unable to easily exit from the mesh; trapped cells may exhibit contact inhibition and cease production of the appropriate factors necessary to support proliferation and maintain long term cultures. If the openings are too large, the stromal cells may be unable to stretch across the opening; this will also decrease stromal cell production of the appropriate factors necessary to support proliferation and maintain long term cultures. When using a mesh type of framework, as exemplified herein, it has been found that openings ranging from about 140 μm to about 220 μm will work satisfactorily. However, depending upon the three-dimensional structure and intricacy of the framework, other sizes may work equally well. In fact, any shape or structure that allows the stromal cells to stretch and continue to replicate and grow for lengthy time periods will work in accordance with the invention.

Different proportions of the various types of collagen deposited on the framework can affect the growth of the cells that come in contact with the 3DFC. The proportions of extracellular matrix (ECM) proteins deposited can be manipulated or enhanced by selecting fibroblasts that elaborate the appropriate collagen type. This can be accomplished using monoclonal antibodies of an appropriate isotype or subclass that are capable of activating complement, and which define particular collagen types. These antibodies and complement can be used to negatively select the fibroblasts which express the desired collagen type. Alternatively, the stroma used to inoculate the framework can be a mixture of cells which synthesize the appropriate collagen types desired. Thus, since the 3DFC described herein is suitable for the growth of diverse cell types and tissues, and depending upon the tissue to be cultured and the collagen types desired, the appropriate stromal cell(s) may be selected to inoculate the three-dimensional framework.

During incubation of the 3DFC, proliferating cells may be released from the framework. These released cells may stick to the walls of the culture vessel where they may continue to proliferate and form a confluent monolayer. This should be prevented or minimized, for example, by removal of the released cells during feeding, or by transferring the 3DFC to a new culture vessel. The presence of a confluent monolayer in the vessel may “shut down” the growth of cells in the 3DFC. Removal of the confluent monolayer or transfer of the stromal tissue to fresh media in a new vessel will restore proliferative activity of the 3DFC. Such removal or transfers should be done in any culture vessel which has a stromal monolayer exceeding 25% confluency. Alternatively, the 3DFC could be agitated to prevent the released cells from sticking, or instead of periodically feeding the cultures, the culture system could be set up so that fresh media continuously flows through the 3DFC. The flow rate could be adjusted to both maximize proliferation within the 3DFC, and to wash out and remove cells released from the culture, so that they will not stick to the walls of the vessel and grow to confluence.

In a second aspect, the present invention provides methods for seeding a three dimensional fibroblast construct (3DFC) with cells, comprising:

(a) contacting a cultured 3DFC with a cell sheet to be seeded onto the 3DFC; and

(b) culturing the cell sheet under conditions suitable for the cell sheet to adhere to the 3DFC.

The inventors have unexpectedly discovered that the methods of this aspect of the invention permit the harvest of cells sheets for seeding onto a 3DFC, and thus permit their use as a delivery method, such as the methods disclosed below for treating chronic heart failure.

In one alternative embodiment, cell sheets with cardiac stem cells are adhered to the 3DFC, allowing cardiac stem cells to establish and maintain cellular/electrical communications such as gap junctions prior to placement onto the myocardium in the methods of the invention for treating CHF, discussed in more detail below.

All terms in this second aspect are as defined for the first aspect of the invention, and all embodiments of the first aspect of the invention are equally applicable to this second aspect of the invention. In this second aspect, the 3DFC is contacted with a cell sheet under conditions suitable for adherence to the 3DFC. As used herein, a “cell sheet” comprises confluent cells dissociated from culture as an intact “sheet” for contacting with the 3DFC. In an alternative embodiment, the disassociating is effected by chilling the culture substrate to a temperature effective to release said cells as an intact cell sheet; any suitable temperature may be used, preferably between 10-20° C.

Contacting a cultured 3DFC with a cell sheet to be seeded onto the 3DFC and subsequent culturing to promote cell sheet adherence to the 3DFC can be done under any suitable conditions, such as those described in the examples that follow, and those discussed with respect to the first aspect of the invention. Based on the teachings herein, those of skill in the art can determine appropriate conditions for contacting the cultured 3DFC with the cell sheet to be seeded onto the 3DFC; and culturing the cell sheet under conditions suitable for the cell sheet to adhere to the 3DFC. In one exemplary alternative embodiment, the cells of interest are grown in culture until confluency, followed by chilling of the culture substrate to approximately 10° C.-20° C.), which allows dissociation of the cell sheet from the culture substrate. Dissociation is generally complete within (30-60 minutes, and the dissociated cell sheet is taken up by any suitable means, such as by appropriately sized pipette and transferred onto the 3DFC. In one alternative embodiment, a suitable amount of culture media (1-5 μl; preferably 2-3 ul) is placed onto the cell sheet so that it unfolds to lay flat against the 3DFC. Any suitable cell media may be used, including but not limited to DMEM supplemented with 5-15% fetal bovine serum (preferably approximately 10%). After the cell sheet is relatively flattened and is flush against the 3DFC excess media is removed using a pipette. The 3DFC cell sheet complex is placed in the incubator for 5 min at 5.0% CO₂ and 37 degrees C. After which, the 3DFC cell sheet complex is re-suspended in 37 degree C. 10% FBS in DMEM and cultured overnight. The cell sheet is preferably centrally placed on the 3DFC, and is placed so as to minimize folds or bunching in the cell sheet. The construct is then incubated under appropriate conditions to promote adhesion of the cells in the cell sheet to the 3DFC. Any suitable conditions can be used. In one alternative embodiment that can be combined with any other embodiments herein, the incubation occurs at 37° C. and 5% CO₂ for between 5 and 15 minutes; more preferably between 7.5 and 12.5 minutes; most preferably approximately 10 minutes.

In a further alternative embodiment, the desired cells are grown and cultured in temperature sensitive plates under standard culture conditions. Once confluent, cell media volume is reduced by 50% and the plates are chilled at 20 degrees C. for 30-60 min or until cell sheets dissociate from the culture plate. Using a pipette, cell sheets are harvested, placed on thawed 3DFC and flattened using droplets of standard culture media. Once flattened, excess media is removed using a pipette. The 3DFC cell sheet complex is placed in the incubator (5.0% CO₂ at 37° C. for 5 min. 3DFC cell sheet complex is re-suspended in 37° C. standard culture medium, placed back in the incubator and cultured over night. Culture media is changed every 48 hrs.

Any suitable initial seeding densities in the temperature sensitive culture plates can be used, and may vary on cell type. In one alternative embodiment, cardiomyocytes or precursors thereof are seeded at approximately 1-2×10⁴ cells/mm²; preferably approximately 1.4×10⁴ cells/mm².

The cells in the cell sheet may be of any desired type, including but not limited to muscle cells (skeletal muscle cells, smooth muscle cells, cardiac muscle cells such as cardiomyocyte) and progenitors thereof, endothelial progenitor cells, bone marrow cells, umbilical cord blood cells, and combinations thereof. In an alternative embodiment cell types with strong cell-cell interactions, such as endothelial cells and cardiomyocytes or progenitors thereof, are used.

In an alternative embodiment of all aspects of the invention, the seeded cells comprise cardiomyocytes and/or progenitors thereof such as cardiac stem cells. There are a limited number of intrinsic cardiac stem cells in the mature adult heart that are self-renewing, clonogenic, and multipotent, such that they differentiate into cardiomyocytes and, to a lesser extent, into smooth muscle and endothelial cells. Cardiac stem cells can be isolated and expanded in culture indefinitely and are characterized by cell surface markers: Lin−, c-Kit+, CD45−, CD34−. See, for example, Messina et al., Circulation Research, 2004, 95:911; Barile et al., Nat. Clin. Prac. Cardiovasc. Med. 2007, February 4, Suppl 1: S9-S14; Noort et al., Pediatric Cardiology, 30(5):699 (2009); Cardiac stem cell isolation kit available from Millipore (Cat. #SCR061)

The cells to be seeded may be recombinant cells capable of expressing a gene product of interest for a given purpose. In one alternative embodiment as described in more detail below, cardiomyocytes engineered to express thymosin beta-4 are seeded onto the 3DFC.

It will be understood by those of skill in the art that it is not a requirement that all cells in the cell sheet contact the 3DFC. In one alternative embodiment, the seeded cell sheets may contact each other, to yield two or more cell layers atop the 3DFC. It will be understood by those of skill in the art that it is not a requirement that all cells in the cell sheet adhere to the 3DFC as a result of the force application, as the cells are preferably present in the suspension in an amount that saturates all available locations for adherence onto the 3DFC. In an alternative embodiment that can be combined with any other embodiment disclosed herein, the cells are adhered to the 3DFC at a cell density ranging between 0.5×10⁶ cells/cm² and 5×10⁶ cells/cm²; more preferably between 1×10⁶ cells/cm² and 4×10⁶ cells/cm²; and most preferably between 1.5×10⁶ cells/cm² and 3×10⁶ cells/cm².

In a further alternative embodiment that can be combined with any of the other embodiments disclosed herein, the culturing further comprises growth of cells adhered to the 3DFC. In this embodiment, such growth can occur under the same or different culture conditions than those used to promote adherence of the cell sheet to the 3DFC. Suitable culture conditions to promote proliferation and/or differentiation of cells adhered to the 3DFC can be determined by those of skill in the art, based on the disclosure herein. Once adhesion of the sheet occurs, the 3DFC cell sheet complex can be cultured under standard culture methods.

In a third aspect, the present invention provides constructs comprising muscle cells adhered to a 3DFC, wherein the cells are capable of spontaneous synchronized contractions across the 3DFC; and wherein the muscle cells are seeded on the construct at a density of between 0.5×10⁶ cells/cm² and 5×10⁶ cells/cm² and/or the muscle cells are present in a ratio of between about 1:10 and about 10:1 with fibroblasts on the construct. Terms in this third aspect of the invention retain the meaning disclosed in the first and second aspects of the invention. All embodiments of the first and second aspects of the invention are equally applicable to this third aspect of the invention, unless the context clearly indicates otherwise. In another embodiment, fibroblasts are present on the construct at a density of between about 5.0×10⁵ cells/cm² and about 5.0×10⁶ cells/cm²; in another embodiment between about 8.0×10⁵ cells/cm² and about 2.0×10⁶ cells/cm².

As discussed in more detailed below, the inventors have discovered that the constructs of this aspect of the invention can be used, for example, in cell therapy for treating various disorders such as chronic heart failure. The inventors have further discovered that the resulting construct provides for cell-cell communication across the construct, which can beat synchronously.

In one alternative embodiment, the 3DFC comprises a patch, with the cells seeded onto a top portion of the patch. In this embodiment, the bottom portion of the patch can be attached to a surface of interest, such as the heart.

The constructs of the invention comprise muscle cells are seeded on the construct at a density of between 0.5×10⁶ cells/cm² and 5×10⁶ cells/cm² and/or muscle cells are present in a ratio about of between 1:10 and about 10:1 with fibroblasts on the construct. In one embodiment, muscle cells are seeded on the construct at a density of between 1.5×10⁶ cells/cm² 3.0×10⁶ cells/cm². In another embodiment, the muscle cells are present in a ratio of between about 1:10 and about 10:1 with fibroblasts on the construct. In another embodiment, the muscle cells are seeded on the construct at a density of between 0.5×10⁶ cells/cm² and 5×10⁶ and the muscle cells are present in a ratio of between 1:10 and 10:1 with fibroblasts on the construct. In a preferred embodiment of all these embodiments, the muscle cells are cardiomyocytes or precursors thereof.

In various embodiments, the muscle cells (such as cardiomyocytes or precursors thereof) are present in a ratio with fibroblasts on the construct of between about 1:10 and about 10:1; about 1:9 to about 9:1; about 1:8 to about 8:1; about 1:7 to about 7:1; about 1:6 to about 6:1; about 1:5 to about 5:1; about 1:4 to about 4:1; about 1:3 to about 3:1; about 1:2 to about 2:1; or about 1:1. Other ratios of cells are also possible. In a preferred embodiment of all these embodiments, the muscle cells are cardiomyocytes or precursors thereof.

In various embodiments, the muscle cells (such as cardiomyocytes or precursors thereof) are seeded on the construct at a density between 1.3×10⁶ cells/cm² and 5×10⁶ cells/cm²; more preferably between 1.4×10⁶ cells/cm² and 4×10⁶ cells/cm²; and most preferably between 1.5×10⁶ cells/cm² and 3×10⁶ cells/cm². In a preferred embodiment of all these embodiments, the muscle cells are cardiomyocytes or precursors thereof

In another embodiment, the cardiomyocytes or precursors thereof are seeded on the construct at a density between 1.5×10⁶ cells/cm² and 3×10⁶ cells/cm², and the fibroblasts are present on the construct at a density of between about 8.0×10⁵ cells/cm² and about 2.0×10⁶ cells/cm².

As used herein, “muscle cells” can be skeletal muscle cells, smooth muscle cells, cardiac muscle cells, or precursors thereof. In an alternative embodiment that can be combined with any other embodiment herein, the muscle cells comprise cardiomyocytes or precursors thereof; most preferably of human origin. In a further alternative embodiment that can be combined with any other embodiment, the construct is a seeded, non-contracting patch, which can be ready for implantation by 24 hours after seeding and used, for example, to provide cardiomyocytes to the host heart. In another alternative embodiment that can be combined with any other embodiment, the construct is a contractile construct, is capable of spontaneous synchronized contractions, and may be used, for example, for contractile assistance. As used herein, the phrase “spontaneous synchronized contractions” means that the cells are capable of producing coordinated force generation across the 3DFC. As used herein, “across the 3DFC” means that the cells are capable of spontaneous synchronized contractions over the full 3DFC where the cells are adhered.

In a further alternative embodiment that can be combined with any other embodiment herein, the muscle cells, or precursors thereof, are obtained from a subject (such as by standard biopsy procedures or other surgical procedures) to be treated with the construct in the methods of the invention described below. In a further alternative embodiment that can be combined with any other embodiment herein, the cells can be engineered to express a protein of interest, such as cardiomyocytes or precursors thereof engineered to express one or more of thymosin beta-4 (TB4), akt murine thyoma viral oncogene homolog (AKT1; SEQ ID NO:1, 3, or 5 for amino acid sequence), stroma cell-derived factor-1 alpha (SDF-1; SEQ ID NO:7 for amino acid sequence), and hepatocyte growth factor (HGF; SEQ ID NO:9 for amino acid sequence), as disclosed above.

The constructs can be made shortly prior (ie, 0-10 days prior) to a desired implantation according to the methods of the invention described below, or may be stored frozen and rethawed prior to implantation.

The muscle cells can be adhered to the 3DFC at any density suitable for a given application. In an alternative embodiment that can be combined with any other embodiment disclosed herein, the muscle cells (such as cardiomyocytes or precursors thereof) are adhered to the 3DFC at a cell density ranging between 1.3×10⁶ cells/cm² and 5×10⁶ cells/cm²; more preferably between 1.4×10⁶ cells/cm² and 4×10⁶ cells/cm²; and most preferably between 1.5×10⁶ cells/cm² and 3×10⁶ cells/cm². Lower density cardiomyocyte seedings (0.6-1.2×10⁶ cells/cm²) displayed non-synchronized yet spontaneous contractions, while higher density constructs disclosed were capable of consistent rhythmic and directional contractions

Specific methods for producing constructs according to this aspect of the invention are provided in the examples that follow. In one embodiment, the methods of any embodiment or combination of embodiments of the first or second aspect of the invention can be used to make the constructs of this third aspect. In one alternative embodiment, cell sheets with cardiac stem cells or cardiomyocytes are adhered to the 3DFC, allowing cardiac stem cells or cardiomyocytes to which they give rise to establish and maintain cellular/electrical communications such as gap junctions prior to placement onto the myocardium in the methods of the invention for treating CHF, discussed in more detail below.

In a fourth aspect, the present invention provides methods for treating a disorder characterized by a lack of functioning cardiomyocytes, comprising contacting the heart of a subject suffering from such a disorder with an amount effective to treat the disorder of a construct according to the third aspect of the invention that comprise cardiomyocytes or precursors thereof adhered to a 3DFC.

The inventors have unexpectedly discovered that the constructs of this aspect of the invention can be used, for example, in cell therapy for treating disorders characterized by a lack of functioning cardiomyocytes. Such disorders include, but are not limited to, chronic heart failure (CHF), ischemia without heart failure, cardiomyopathy (such as dilated cardiomyopathy (DCM)), cardiac arrest, congestive heart failure, stable angina, unstable angina, myocardial infarction, coronary artery disease, valvular heart disease, ischemic heart disease, reduced ejection fraction, reduced myocardial perfusion, maladaptive cardiac remodeling (such as left ventricle remodeling), reduced left ventricle function, left heart failure, right heart failure, backward heart failure (increased venous back pressure), forward heart failure (failure to supply adequate arterial perfusion), systolic dysfunction, diastolic dysfunction, systemic vascular resistance, low-output heart failure, high-output heart failure, dyspnea on exertion, dyspnea at rest, orthopnea, tachypnea, paroxysmal nocturnal dyspnea, dizziness, confusion, cool extremities at rest, exercise intolerance, easy fatigueability, peripheral edema, nocturia, ascites, hepatomegaly, pulmonary edema, cyanosis, laterally displaced apex beat, gallop rhythm, heart murmurs, parasternal heave, and pleural effusion. In one embodiment the disorder is CHF; in another embodiment the disorder is DCM. In another embodiment, the disorder is ischemia without heart failure. While not being bound by any mechanism of action, the inventors believe that current failure of cell therapy efforts to treat disorders such as CHF (based at least in part on the lack of survival of implanted stem cells) is related to trying to grow cells in a hostile environment without adequate blood supply/matrix support. Without adequate extracellular matrix, injected cells clump due to lack of physical support for the cells to attach. In contrast, co-populating the 3DFC with cardiomyocytes and/or cardiac stem cells as per the methods of the present invention will enable these cells to grow and engraft onto the heart. This coupled with improved blood flow will result in more viable myocardium to treat the disorders disclosed above, for example, through improved left ventricular (LV) function and reduced maladaptive LV remodeling. The 3DFC provides growth factor stimulation to enhance matrix support/new blood vessel formation allowing cardiomyocytes and/or cardiac stem cells to engraft and grow. Fibroblasts in the 3DFC produce angiogenic growth factors: VEGF (vascular endothelial growth factor), HGF (hepatocyte growth factor), bFGF (basic fibroblast growth factor), and angiopoietin-1 (See, for example, J. Anat. (2006) 209, pp 527-532), which also help to provide vasculature for survival of the seeded cardiomyocytes. Thus, a significant advantage of the co-culture on 3DFC is that the 3DFC is pro-angiogenic or pro-arteriogenic, and thus addresses ischemia at the same time the co-cultured patch is delivering cells or beating function to the heart. Thus, the underlying 3DFC makes the ischemic myocardium more friendly to the seeded cells and thus more likely to survive and become functional, which can include migrating off the construct to functionally integrate into the myocardium, or remaining on the construct. As will be clear to those of skill in the art, the construct also can be used as an adjunct therapy, to provide a pumping assist without integration of the cardiomyocytes into the heart myocardium.

Thus, in a further alternative embodiment that can be combined with any other embodiment herein, the construct is a seeded, non-contracting 3DFC patch to provide cardiomyocytes to the host heart and to become functional or to functionally integrate into the myocardium. In another alternative embodiment that can be combined with any other embodiment herein, the construct is a contractile construct and may be used, for example, as an adjunct therapy, to provide a pumping assist without integration of the cardiomyocytes into the heart myocardium.

In the methods of the invention, attaching the matrix scaffold to the heart subjects it to mechanical stress that stimulates cell migration, growth, and secretion of extracellular matrix protein. The rhythmic stretching of the scaffold facilitates nutrient and waste exchange within the scaffold by opening and compressing the scaffold pores.

Thus, the present methods utilize the 3DFC as a delivery system for cell-based therapy using the heart as its own bioreactor to support the engraftment/growth of cells seeded on the 3DFC. The methods of the invention permit covering a larger amount of myocardium as opposed to isolated cell injections, thus addressing one criticism as to why cell injections appear to work better in rodents than humans, ie., the amount of damaged myocardium needed to treat. Also cells seeded on the 3DFC will not wash out in the circulation as seen with insolated cell injections.

In an alternative embodiment that can be combined with any other embodiments herein, the subject is a mammal, most preferably a human. In a further alternative embodiment that can be combined with any other embodiments herein, the subject is human. In another alternative embodiment, the cardiomyocytes or cardiac stem cells are obtained from the subject.

As used herein, “CHF” is a chronic (as opposed to rapid onset) impairment of the heart's ability to supply adequate blood to meet the body's needs. CHF may be caused by, but is distinct from, cardiac arrest, myocardial infarction, and cardiomyopathy. In one alternative embodiment, the subject suffers from congestive heart failure. In various further alternative embodiments that can be combined with any other embodiments herein, the subject's heart failure comprises left heart failure, right heart failure, backward heart failure (increased venous back pressure), forward heart failure (failure to supply adequate arterial perfusion), systolic dysfunction, diastolic dysfunction, systemic vascular resistance, low-output heart failure, high-output heart failure. In various further alternative embodiments that can be combined with any other embodiments herein, the subject's CHF may be any of Classes I-IV as per the New York Heart Association Functional Classification; more preferably Class III or IV.

-   -   Class I: no limitation is experienced in any activities; there         are no symptoms from ordinary activities.     -   Class II: slight, mild limitation of activity; the patient is         comfortable at rest or with mild exertion.     -   Class III: marked limitation of any activity; the patient is         comfortable only at rest.     -   Class IV: any physical activity brings on discomfort and         symptoms occur at rest.

In a further alternative embodiment that can be combined with any other embodiments herein, the subject has been diagnosed with CHF according to the New York Heart Association Functional Classification. In a further alternative embodiment that can be combined with any other embodiments herein, the subject is further characterized by one or more of the following: hypertension, obesity, cigarette smoking, diabetes, valvular heart disease, and ischemic heart disease.

As used herein, “treat” or “treating” means accomplishing one or more of the following: (a) reducing the severity of the disorder (ex: treatment of Class IV subject to improve status to Class III for CHF subjects); (b) limiting or preventing development of symptoms characteristic of the disorder; (c) inhibiting worsening of symptoms characteristic of the disorder; (d) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorder. Signs characteristic of CHF include, but are not limited to reduced ejection fraction, reduced myocardial perfusion, maladaptive cardiac remodeling (such as left ventricle remodeling), reduced left ventricle function, dyspnea on exertion, dyspnea at rest, orthopnea, tachypnea, paroxysmal nocturnal dyspnea, dizziness, confusion, cool extremities at rest, exercise intolerance, easy fatigueability, peripheral edema, nocturia, ascites, hepatomegaly, pulmonary edema, cyanosis, laterally displaced apex beat, gallop rhythm, heart murmurs, parasternal heave, and pleural effusion.

In one embodiment, the constructs described herein find use in promoting the healing of ischemic heart tissue. The ability of the constructs to promote the healing of an ischemic tissue depends in part, on the severity of the ischemia. As will be appreciated by the skilled artisan, the severity of the ischemia depends, in part, on the length of time the tissue has been deprived of oxygen. Among such activities is the reduction or prevention of the remodeling of ischemic tissue. By “remodeling” herein is meant, the presence of one or more of the following: (1) a progressive thinning of the ischemic tissue, (2) a decrease in the number or blood vessels supplying the ischemic tissue, and/or (3) a blockage in one or more of the blood vessels supplying the ischemic tissue, and if the ischemic tissue comprises muscle tissue, (4) a decrease in the contractibility of the muscle tissue. Untreated, remodeling typically results in a weakening of the ischemic tissue such that it can no longer perform at the same level as the corresponding healthy tissue. Cardiovascular ischemia is generally a direct consequence of coronary artery disease, and is usually caused by rupture of an atherosclerotic plaque in a coronary artery, leading to formation of thrombus, which can occlude or obstruct a coronary artery, thereby depriving the downstream heart muscle of oxygen. Prolonged ischemia can lead to cell death or necrosis, and the region of dead tissue is commonly called an infarct.

In some embodiments, candidate subjects for the methods described herein will be patients with stable angina and reversible myocardial ischemia. Stable angina is characterized by constricting chest pain that occurs upon exertion or stress, and is relieved by rest or sublingual nitroglycerin. Coronary angiography of patients with stable angina usually reveals 50-70% obstruction of at least one coronary artery. Stable angina is usually diagnosed by the evaluation of clinical symptoms and ECG changes. Patients with stable angina may have transient ST segment abnormalities, but the sensitivity and specificity of these changes associated with stable angina are low.

In some embodiments, candidates for the methods described herein will be patients with unstable angina and reversible myocardial ischemia. Unstable angina is characterized by constricting chest pain at rest that is relieved by sublingual nitroglycerin. Anginal chest pain is usually relieved by sublingual nitroglycerin, and the pain usually subsides within 30 minutes. There are three classes of unstable angina severity: class I, characterized as new onset, severe, or accelerated angina; class II, subacute angina at rest characterized by increasing severity, duration, or requirement for nitroglycerin; and class III, characterized as acute angina at rest. Unstable angina represents the clinical state between stable angina and acute myocardial infarction (AMI) and is thought to be primarily due to the progression in the severity and extent of atherosclerosis, coronary artery spasm, or hemorrhage into non-occluding plaques with subsequent thrombotic occlusion. Coronary angiography of patients with unstable angina usually reveals 90% or greater obstruction of at least one coronary artery, resulting in an inability of oxygen supply to meet even baseline myocardial oxygen demand. Slow growth of stable atherosclerotic plaques or rupture of unstable atherosclerotic plaques with subsequent thrombus formation can cause unstable angina. Both of these causes result in critical narrowing of the coronary artery. Unstable angina is usually associated with atherosclerotic plaque rupture, platelet activation, and thrombus formation. Unstable angina is usually diagnosed by clinical symptoms, ECG changes, and changes in cardiac markers.

In some embodiments, candidates for the methods described herein will be human patients with left ventricular dysfunction and reversible myocardial ischemia that are undergoing a coronary artery bypass graft (CABG) procedure, who have at least one graftable coronary vessel and at least one coronary vessel not amenable to bypass or percutaneous coronary intervention.

In some embodiments, application of the construct to an ischemic tissue increases the number of blood vessels present in the ischemic tissue, as measured using laser Doppler imaging (see, e.g., Newton et al., 2002, J Foot Ankle Surg, 41(4):233-7). In some embodiments, the number of blood vessels increases 1%, 2%, 5%; in other embodiments, the number of blood vessels increases 10%, 15%, 20%, even as much as 25%, 30%, 40%, 50%; in some embodiments, the number of blood vessels increase even more, with intermediate values permissible.

In some embodiments, application of the construct to an ischemic heart tissue increases the ejection fraction. In a healthy heart, the ejection fraction is about 65 to 95 percent. In a heart comprising ischemic tissue, the ejection fraction is, in some embodiments, about 20-40 percent. Accordingly, in some embodiments, treatment with the construct results in a 0.5 to 1 percent absolute improvement in the ejection fraction as compared to the ejection fraction prior to treatment. In other embodiments, treatment with the construct results in an absolute improvement in the ejection fraction more than 1 percent. In some embodiments, treatment results in an absolute improvement in the ejection fraction of 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, or more as compared to the ejection fraction prior to treatment. For example, if the ejection fraction prior to treatment was 40%, then following treatment ejection fractions between 41% to 59% or more are observed in these embodiments. In still other embodiments, treatment with the construct results in an improvement in the ejection fraction greater than 10% as compared to the ejection fraction prior to treatment.

In some embodiments, application of the construct to an ischemic heart tissue increases one or more of cardiac output (CO) (increases of up to 55% or more relative to pre-status treatment), left ventricular end diastolic volume index (LVEDVI), left ventricular end systolic volume index (LVESVI), and systolic wall thickening (SWT). These parameters are measured by art-standard clinical procedures, including, for example, nuclear scans, such as radionuclide ventriculography (RNV) or multiple gated acquisition (MUGA), and X-rays.

In some embodiments, application of the construct to an ischemic heart tissue causes a demonstrable improvement in the blood level of one or more protein markers used clinically as indicia of heart injury, such as creatine kinase (CK), serum glutamic oxalacetic transaminase (SGOT), lactic dehydrogenase (LDH) (see, e.g., U.S. Publication 2005/0142613), troponin I and troponin T can be used to diagnose heart muscle injury (see, e.g., U.S. Publication 2005/0021234). In yet other embodiments, alterations affecting the N-terminus of albumin can be measured (see, e.g., U.S. Publications 2005/0142613, 2005/0021234, and 2005/0004485; the disclosures of which are incorporated herein by reference in their entireties).

Additionally, the cultured three-dimensional tissue can be used with therapeutic devices used to treat heart disease including heart pumps, endovascular stents, endovascular stent grafts, left ventricular assist devices (LVADs), biventricular cardiac pacemakers, artificial hearts, and enhanced external counterpulsation (EECP).

In a further alternative embodiment that can be combined with any other embodiments herein, the treating results in production of new cardiomyocytes and new blood vessels in the subject. In a further alternative embodiment that can be combined with any other embodiments herein, the treating results in improvement of left ventricular function, fall in end diastolic pressure (EDP) (reduction of up to 50-60% or more relative to pre-status treatment), myocardial perfusion, repopulation of the anterior wall with cardiomyocytes, and/or reversing maladaptive left ventricle remodeling in the subject.

In one non-limiting alternative embodiment in which a synchronously beating construct is placed on the heart to aid in contraction of the left ventricle, beneficial treatment can be demonstrated by an improvement in ejection fraction. In a further non-limiting alternative embodiment, a non-beating construct is placed on the heart, then spontaneously begins beating on the heart to aid in contraction of the heart.

The construct can be contacted with the heart in any suitable way to promote attachment. The construct may be attached to various locations on the heart, including the epicardium, myocardium and endocardium, most preferably the epicardium. Means for attachment include, but are not limited to, direct adherence between the construct and the heart tissue, biological glue, suture, synthetic glue, laser dyes, or hydrogel. A number of commercially available hemostatic agents and sealants include SURGICAL® (oxidized cellulose), ACTIFOAM® (collagen), FIBRX® (light-activated fibrin sealant), BOHEAL® (fibrin sealant), FIBROCAPS® (dry powder fibrin sealant), polysaccharide polymers p-GlcNAc (SYVEC® patch; Marine Polymer Technologies), Polymer 27CK (Protein Polymer Tech.). Medical devices and apparatus for preparing autologous fibrin sealants from 120 ml of a patient's blood in the operating room in one and one-half hour are also known (e.g. Vivostat System).

In an alternative embodiment of the invention utilizing direct adherence, the construct is placed directly onto the heart and the product attaches via natural cellular attachment. In a further alternative embodiment, the construct is attached to the heart using surgical glue, preferably biological glue such as a fibrin glue. The use of fibrin glue as a surgical adhesive is well known. Fibrin glue compositions are known (e.g., see U.S. Pat. Nos. 4,414,971; 4,627,879 and 5,290,552) and the derived fibrin may be autologous (e.g., see U.S. Pat. No. 5,643,192). The glue compositions may also include additional components, such as liposomes containing one or more agent or drug (e.g., see U.S. Pat. Nos. 4,359,049 and 5,605,541) and include via injection (e.g., see U.S. Pat. No. 4,874,368) or by spraying (e.g., see U.S. Pat. Nos. 5,368,563 and 5,759,171). Kits are also available for applying fibrin glue compositions (e.g., see U.S. Pat. No. 5,318,524).

In another embodiment, a laser dye is applied to the heart, the construct, or both, and activated using a laser of the appropriate wavelength to adhere to the tissues. In alternative embodiments, the laser dye has an activation frequency in a range that does not alter tissue function or integrity. For instance, 800 nm light passes through tissues and red blood cells. Using indocyan green (ICG) as the laser dye, laser wavelengths that pass through tissue may be used. A solution of 5 mg/ml of ICG is painted onto the surface of the three-dimensional stromal tissue (or target site) and the ICG binds to the collagen of the tissue. A 5 ms pulse from a laser emitting light with a peak intensity near 800 nm is used to activate the laser dye, resulting in the denaturation of collagen which fuses elastin of the adjacent tissue to the modified surface.

In another embodiment, the construct is attached to the heart using a hydrogel. A number of natural and synthetic polymeric materials are sufficient for forming suitable hydrogel compositions. For example, polysaccharides, e.g., alginate, may be crosslinked with divalent cations, polyphosphazenes and polyacrylates are crosslinked ionically or by ultraviolet polymerization (U.S. Pat. No. 5,709,854). Alternatively, a synthetic surgical glue such as 2-octyl cyanoacrylate (“DERMABOND”, Ethicon, Inc., Somerville, N.J.) may be used to attach the three-dimensional stromal tissue.

In an alternative embodiment of the present invention, the construct is secured to the heart using one or more sutures, including, but not limited to, 5-O, 6-O and 7-O proline sutures (Ethicon Cat. Nos. 8713H, 8714H and 8701H), poliglecaprone, polydioxanone, polyglactin or other suitable non-biodegradable or biodegradable suture material. When suturing, double armed needles are typically, although not necessarily, used.

In another embodiment, the 3DFC is grown in a bioreactor system (e.g., U.S. Pat. Nos. 5,763,267 and 5,843,766) in which the framework is slightly larger than the final tissue-engineered product. The final product contains a border, one edge, flap or tab of the scaffold material, which is used as the site for application of the biological/synthetic glue, laser dye or hydrogel. In alternative embodiments, the scaffold weave may be used as an attachment for suturing or microsuturing.

As used herein, the phrase “an amount effective” means an amount of the construct that will be effective to treat the disorder, as discussed herein. As will be clear to those of skill in the art, the methods comprise the use of one or more of the recited constructs to treat disorders characterized by a lack of functioning cardiomyocytes. In one embodiment, the method comprises contacting the heart with an amount of one or more constructs that serves to cover one or more ischemic regions of the heart, preferably all ischemic regions of the heart. The construct is used in an amount effective to promote tissue healing and/or revascularization of weakened or damaged heart tissue in an individual diagnosed with a disorder characterized by a lack of functioning cardiomyocytes. The amount of the construct administered, depends, in part, on the severity of the disorder, whether the construct is used as an injectable composition (see, US20060154365, incorporated herein by reference in its entirety), the concentration of the various growth factors and/or Wnt proteins present, the number of viable cells comprising the construct, and/or ease of access to the heart tissue(s) being treated. Determination of an effective dosage is well within the capabilities of those skilled in the art. Suitable animal models, such as the canine model described in US 20060292125 (incorporated by reference herein in its entirety) can be used for testing the efficacy of the dosage on a particular tissue of the heart.

As used herein “dose” refers to the number of cohesive pieces of construct applied to the heart of an individual diagnosed with congestive heart failure. A typical cohesive piece of construct is approximately 35 cm². As will be appreciated by those skilled in the art, the absolute dimensions of the cohesive piece can vary, as long it comprises a sufficient number of cells to promote healing of weakened or damaged heart tissue in an individual diagnosed with a disorder characterized by a lack of functioning cardiomyocytes. Thus, cohesive pieces suitable for use in the methods described herein can range in size from 15 cm² to 50 cm².

The application of more than one cohesive piece of construct can be used to increase the area of the heart treatable by the methods described herein. For example, in embodiments using a two pieces of cohesive construct, the treatable area is approximately doubled in size. In embodiments using three cohesive pieces of construct, the treatable area is approximately tripled in size. In embodiments using four cohesive pieces of construct, the treatable area is approximately quadrupled in size. In embodiments using five cohesive pieces of construct, the treatable area is approximately five-fold, i.e. from 35 cm² to 175 cm².

In some embodiments, one cohesive piece of construct is attached to a region of the heart in an individual diagnosed with a disorder characterized by a lack of functioning cardiomyocytes.

In other embodiments, two cohesive pieces of construct are attached to a region of the heart in an individual diagnosed with a disorder characterized by a lack of functioning cardiomyocytes.

In other embodiments, three cohesive pieces of construct are attached to a region of the heart in an individual diagnosed with a disorder characterized by a lack of functioning cardiomyocytes.

In other embodiments, four, five, or more cohesive pieces of construct are attached to a region of the heart in an individual diagnosed with a disorder characterized by a lack of functioning cardiomyocytes.

In embodiments in which two or more cohesive pieces of construct are administered, the proximity of one piece to another can be adjusted, depending in part on, the severity of the disorder characterized by a lack of functioning cardiomyocytes, the extent of the area being treated, and/or ease of access to the heart tissue(s) being treated. For example, in some embodiments, the pieces of 3DFC can be located immediately adjacent to each other, such that one or more edges of one piece contact one or more edges of a second piece. In other embodiments, the pieces can be attached to the heart tissue such that the edges of one piece do not touch the edges of another piece. In these embodiments, the pieces can be separated from each other by an appropriate distance based on the anatomical and/or disease conditions presented by the subject. Determination of the proximity of one piece to another, is well within the capabilities of those skilled in the art, and if desired can be tested using suitable animal models, such as the canine model described in US20060292125.

In embodiments that comprise a plurality of pieces of construct, some, or all of the pieces can be attached to the same or different areas of the heart.

In embodiments that comprise a plurality of pieces of construct, the pieces are simultaneously attached, or concurrently attached to the heart.

In some embodiments, the construct pieces are administered over time. The frequency and interval of administration depends, in part, on the severity of the disorder, whether the 3DFC is used as an injectable composition (see, US20060154365, incorporated herein by reference in its entirety), the concentration of the various growth factors and/or Wnt proteins present, the number of viable cells comprising the 3DFC, and/or ease of access to the heart tissue(s) being treated. Determination of the frequency of administration and the duration between successive applications is well within the capabilities of those skilled in the art, and if desired, can be tested using suitable animal models, such as the canine model described in US20060292125.

In a further alternative embodiment, one or more construct is contacted with the left ventricle. In a further alternative embodiment, the one or more constructs cover the entire heart.

In embodiments that comprise a plurality of pieces of construct, some, or all of the pieces can be attached to the area comprising the heart. In other embodiments, one or more of the construct pieces can be attached to areas that do not comprise damaged myocardium. For example, in some embodiments, one piece can be attached to an area comprising ischemic tissue and a second piece can be attached to an adjacent area that does not comprise ischemic tissue. In these embodiments, the adjacent area can comprise damaged or defective tissue. “Damaged,” or “defective” tissue as used herein refer to abnormal conditions in a tissue that can be caused by internal and/or external events, including, but not limited to, the event that initiated the ischemic tissue. Other events that can result in ischemic, damaged or defective tissue include disease, surgery, environmental exposure, injury, aging, and/or combinations thereof.

In embodiments that comprise a plurality of pieces of cultured three-dimensional tissue, the construct pieces can be simultaneously attached, or concurrently attached to an ischemic tissue.

In an alternative embodiment that can be combined with any other embodiment disclosed herein, the cardiomyocytes or precursors thereof are adhered to the 3DFC at a cell density ranging between 0.5×10⁶ cells/cm² and 5×10⁶ cells/cm²; more preferably between 1×10⁶ cells/cm² and 4×10⁶ cells/cm²; and most preferably between 1.5×10⁶ cells/cm² and 3×10⁶ cells/cm². In another embodiment that can be combined with any other embodiment disclosed herein, the cardiomyocytes or precursors thereof are seeded on the construct at a density between 1.5×10⁶ cells/cm² and 3×10⁶ cells/cm², and the fibroblasts are present on the construct at a density of between about 8.0×10⁵ cells/cm² and about 2.0×10⁶ cells/cm².

In various embodiments, the cardiomyocytes or precursors thereof are present in a ratio with fibroblasts on the construct of between about 1:10 and about 10:1; about 1:9 to about 9:1; about 1:8 to about 8:1; about 1:7 to about 7:1; about 1:6 to about 6:1; about 1:5 to about 5:1; about 1:4 to about 4:1; about 1:3 to about 3:1; about 1:2 to about 2:1; or about 1:1. Other ratios of cells are also possible.

In a further alternative embodiment that can be combined with any other embodiment herein, the cardiomyocytes or precursors thereof are engineered to express a therapeutic of interest, such as thymosin beta-4 (TB4), akt murine thyoma viral oncogene homolog (AKT1), stroma cell-derived factor-1 alpha (SDF-1), and hepatocyte growth factor (HGF). Alternatively, one or more of thymosin beta-4 (TB4), akt murine thyoma viral oncogene homolog (AKT1), stroma cell-derived factor-1 alpha (SDF-1), and hepatocyte growth factor (HGF) can be added exogenously to the construct prior to implantation. (RegeneRx Biopharmaceuticals, Inc. (Bethesda, Md.) In this embodiment, the cardiomyocytes or precursors thereof will provide new cardiomyocytes to grow onto the damaged heart and the added factor(s) will augment cell migration and engraftment.

In various alternative embodiments, the cardiomyocytes or progenitors thereof are selected from the group consisting of cardiomyocytes, cardiac stem cells (such as c-kit+ cardiac stem cells, CD34+ endothelial progenitor cells, autologous bone marrow cells, and mesenchymal stem cells. In an alternative embodiment that can be combined with any other embodiment disclosed herein, the cardiomyocytes or precursors thereof are human cells, more preferably human cells derived from (ie, obtained from and possibly expanded ex vivo prior to administration) the subject to be treated (such as by standard biopsy or other surgical procedures).

The methods may further comprise systemic administration of cytokines to the subject, including but not limited to Insulin like growth factor (IGF), Hepatic Growth Factor (HGF), and Stromal cell-derived factor a (SDF-1a).

The methods and compositions described herein can be used in combination with conventional treatments, such as the administration of various pharmaceutical agents and surgical procedures. For example, in some embodiments, the cultured three-dimensional tissue is administered with one or more of the medications used to treat a disorder characterized by a lack of functioning cardiomyocytes. Medications suitable for use in the methods described herein include angiotensin-converting enzyme (ACE) inhibitors (e.g., enalapril, lisinopril, and captopril), angiotensin II (A-II) receptor blockers (e.g., losartan and valsartan), diuretics (e.g., bumetanide, furosemide, and spironolactone), digoxin, beta blockers, and nesiritide.

Additionally, the constructs can be used with other options used to treat a disorder characterized by a lack of functioning cardiomyocytes, including heart pumps, also referred to as left ventricular assist devices (LVADs), biventricular cardiac pacemakers, cardiac wrap surgery, artificial hearts, and enhanced external counterpulsation (EECP), and cardiac wrap surgery (see, e.g., U.S. Pat. Nos. 6,425,856, 6,085,754, 6,572,533, and 6,730,016, the contents of which are incorporated herein by reference).

In some embodiments, the construct is used in conjunction with cardiac wrap surgery. In these embodiments, a flexible pouch or jacket is used to deliver and/or attach the construct, which can be placed inside or embedded within the pouch prior to placement over the damaged or weakened heart tissue. In other embodiments, the pouch and the 3DFC can be joined together. For example, the pouch and the construct can be joined together using a stretchable stitch assembly. In other embodiments, the construct can be configured to comprise threads useful for joining the framework to the pouch. U.S. Pat. Nos. 6,416,459, 5,702,343, 6,077,218, 6,126,590, 6,155,972, 6,241,654, 6,425,856, 6,230,714, 6,241,654, 6,155,972, 6,293,906, 6,425,856, 6,085,754, 6,572,533, and 6,730,016 and U.S. Patent Publication Nos. 2003/0229265, and 2003/0229261, the contents of which are incorporated herein by reference, describe various embodiments of pouches and jackets, e.g., cardiac constraint devices, that can be used to deliver and/or attach the construct.

In some embodiments, other devices, in addition to the construct are attached to the pouch, e.g., electrodes for defibrillation, a tension indicator for indicating when the jacket is adjusted on the heart to a desired degree of tensioning, and used in the methods and compositions described herein. See, e.g., U.S. Pat. Nos. 6,169,922 and 6,174,279, the contents of which are incorporated herein by reference.

A number of methods can be used to measure changes in the functioning of the heart in subjects before and after attachment of the construct. For example, an echocardiogram can be used to determine the capacity at which the heart is pumping. The percentage of blood pumped out of the left ventricle with each heartbeat is referred to as the ejection fraction. In a healthy heart, the ejection fraction is about 60 percent. In an individual with chronic heart failure caused by the inability of the left ventricle to contract vigorously, i.e., systolic heart failure, the ejection fraction is usually less than 40 percent. Depending on the severity and cause of the heart failure, ejection fractions typically range from less than 40 percent to 15 percent or less. An echocardiogram can also be used to distinguish between systolic heart failure and diastolic heart failure, in which the pumping function is normal but the heart is stiff.

In some embodiments, echocardiograms are used to compare the ejection fractions before and following treatment with the construct. In certain embodiments, treatment with the construct results in improvements in the ejection fraction between 3 to 5 percent. In other embodiments, treatment with the construct results in improvements in the ejection fraction between 5 to 10 percent. In still other embodiments, treatment with the construct results in improvements in the ejection fraction greater than 10 percent.

Nuclear scans, such as radionuclide ventriculography (RNV) or multiple gated acquisition (MUGA) scanning can be used to determine how much blood the heart pumps with each beat. These tests are done using a small amount of dye injected in the veins of an individual A special camera is used to detect the radioactive material as it flows through the heart. Other tests include X-rays and blood tests. Chest X-rays can be used to determine the size of the heart and if fluid has accumulated in the lungs. Blood tests can be used to check for a specific indicator of congestive heart failure, brain natriuretic peptide (BNP). BNP is secreted by the heart in high levels when it is overworked. Thus, changes in the level of BNP in the blood can be used to monitor the efficacy of the treatment regime.

In a further aspect, the present invention provides kits for treating CHF, comprising a suitable construct as disclosed above and a means for attaching the construct to the heart or organ. The means for attachment may include any such attachment device as described above, for example, a composition of surgical glue, hydrogel, or preloaded prolene needles for microsuturing.

Examples Example 1 3DFC Seeding and Co-Culture

The 3DFC patch is a cryopreserved human fibroblast-derived tissue composed of fibroblasts, extracellular matrix, and a bioabsorbable scaffold (21,22). The fibroblast cells were from a qualified cell bank; tested for animal viruses, retroviruses, cell morphology, karology, isoenzymes, and tumorgenicity, free from viruses, retroviruses, endotoxins and mycoplasma. The 3DFC was supplied frozen (5 cm×7.5 cm); it at −75°±10° C. until ready for use when it was placed in sterile PBS (34-37° C.) and applied to the heart within 60 minutes of removal.

Random Seeding:

The 3DFC (ANGINERA™ obtained from and thawed per Theregen, Inc. protocols) was cut into near circular sections approximately 1.5 cm in diameter to fill the well space of a 24 well culture plate. After the 3DFC was placed in the bottom of the well and completely covering its base, 1.5 ml of media (10% FBS in DMEM-LG) containing >3×10⁶ enothelial progenitor cells (rat neonatal cardiomyocytes) were added in suspension over the 3DFC at 27° C. The plate containing the 3DFC and cellular suspension was then centrifuged at 1300 rpms for 5 min forcing the cells in suspension into the 3DFC at 27° C. The plate containing the 3DFC was then transferred to a cell incubator and incubated 24 hrs at 37° C. and 5% CO₂ to allow further cellular adhesion and proliferation (FIG. 1).

Adhesion of the desired cells onto the 3DFC was greatly enhanced when >3×10⁸ cells/ml are added in suspension. At this concentration there was a “traffic jam effect” that takes place causing all the cells in suspension to come in contact with the 3DFC at the same time thus clogging any openings in the 3DFC not allowing the added cells to pass thru.

Starting out with a large number of cells allowed all cells in suspension to migrate unto the 3DFC, the more cells that make contact with the 3DFC enabled some cells to pass through the pores ending up with 1.5 to 2 million cells seeded on the patch.

Cell Sheet Seeding:

Endothelial progenitor cells (bovine pulmonary arterial endothelial cells (BPAECS)) were grown in 24 well or 6 well culture plate (CellSeed, Japan) until they were confluent. The culture plates were then chilled to 20° C. allowing disassociation of the cell sheet from the culture plate. After 40 min the sheet was fully disassociated (FIGS. 2 & 3), and using a 5 ml, 10 ml, or 1000 ul pipette the disassociated cell sheet was taken up in existing culture media and transferred directly onto the 3DFC (obtained from and thawed per Theregen Inc. protocols).

Using 2-3 ul droplets of culture media dripped onto the new cell sheet (at 27° C.), the cell sheet unfolded to lay flat against the 3DFC. Once flat and flush against the 3DFC, the cell sheet+3DFC complex was incubated at 37° C. and 5% CO₂ for 10 min without additional media allowing for full adhesion of the cell sheet to the 3DFC. After, the seeded 3DFC was incubated over night prior to use (implantation, etc.).

Harvested cells sheets retained all cellular adhesion molecules and self-adhered when placed on the 3DFC. In addition, this method of cell harvesting kept intact all cell-to-cell interactions.

Example 2 Construction of a Spontaneously Contracting Biologically Active Cardiomyocyte Scaffold

Methods:

Cardiomyocytes were isolated from neonatal rats 1-2 days old and seeded onto 3DFC scaffolds that were cut into pieces of approximately 1.5-1.7 cm² in diameter. (ANGINERA™, obtained from Theregen, Inc) at concentrations ranging from 0.6×10⁶ to 2.7×10⁶ cells/cm². Briefly, the hearts were excised, atria removed and ventricles cut into 0.5-1 mm portions, minced, then digested in a pancreatin/collagenase solution. Following each enzymatic digest, cardiomyocytes were collected, combined and re-suspended in DMEM with 10% FBS. Lastly, the suspension was differentially plated in Ham's F-12 with 100 mg/ml BSA. The neonatal cardiomyocyte-3DFC were cultured between 1-10 days at 37° C. with 5% CO₂ in 10% FBS in DMEM-LG. Media was changed 24 hrs after initial plating then every 48 hrs.

Results:

Higher density cardiomyocyte seedings (1.8×10⁶ to 2.7×10⁶ cells/cm²) of the 3DFC showed synchronized and spontaneous contractions of the entire scaffold after 48 hrs in culture. Contractions increased in robustness from 48 hrs to 5 days. Lower density cardiomyocyte seedings (0.6-1.2×10⁶ cells/cm²) displayed non-synchronized yet spontaneous contractions. At 72 hours these contractions began synchronizing and by 84 hours, cell contractions were fully synchronized but contracted in a non-consistent manner. At 5 days, scaffolds seeded with 2.7×10⁶ cells/cm² contracted in a consistent rhythmic and directional fashion (ie: medially, with each patch beating in a repetitive directional motion, squeezing “inwards”). Contractions were recorded at 71±3 beats BPM with a mean displacement of 2.9±0.1 mm and contraction velocity of 3.4±0.5 mm/sec (N=10).

CONCLUSION

These findings showed that isolated cardiomyocytes can be seeded and co-cultured onto a biodegradable 3-dimensional construct in a manner allowing cellular survival, communication and electrical coupling. These claims are supported by the observation that the newly seeded neonatal cardiomyocyte-3DFC scaffolds beat spontaneously and in a synchronized, directional fashion with no electrical stimulation. This newly formed neonatal cardiomyocyte 3DFC scaffold is a new and unique cell delivery system to treat CHF.

Example 3 Cellular Communications/Alignment

This example describes cardiomyocyte alignment on cardiomyocyte seeded 3DFC patches and in vivo improvements in rats with heart failure with treatment using the described cardiomyocyte seeded 3DFC (NCM-3DFC).

Connexin Formation:

As shown above, isolated cardiomyocytes can be seeded and co-cultured onto the 3DFC; that these seeded patches contract spontaneously and rhythmically. In theory, rhythmic contractions support the hypothesis that the seeded cardiomyocytes align and establish complex connections with each other allowing for electromechanical cellular communication. These connections, known as connexins, are essential for the rhythmic contraction of cardiac muscle.

Cellular Injections:

The following example shows connexin formation 24 hrs after cardiomycocyte seeding (same cell type as in example 2) and increased cellular connectiveness through connexin formation over time, up to 6 days, thus demonstrating connexin functionality within the 3DFC. In short, dye transfer was evaluated in both 3DFC and NCM-3DFC cultured for 6 days. Three dyes were injected simultaneously: [2-(4-nitro-2,1,3-benzoxadiol-7-yl)aminoethyl]trimethylammonium (NDB-TMA; mol wt 280, net charge 1+, 10 mM), Alexa 350 (mol wt 326, net charge 1−, 10 mM), and Rhodamine Dextran (0.1 mg/ml). Microelectrode tips were created from 1.0 mm filament glass (A-M Systems, Everett, Wash.) on a Sutter Instruments puller (Novato, Calif.). Tips were filled by capillary action with a mixture of the three dyes, backfilled with 200 mM KCl, and then lowered onto cell surface. Dye was slowly and continuously injected by capacitance overcompensation of the amplifier (A-M Systems). Photos were taken every 30 seconds (up to 2.5 min) then every 2.5 minutes up to 20 minutes. Cells that received dye from the injected cell after 10 minutes were compared between groups. Injections into cardiomyocyte seeded patches resulted in extensive dye passage between seeded cardiomyocytes (FIG. 4), demonstrating the functional presence of connexins. Non-seeded patches (fibroblasts only) resulted in retention of dye strictly in the primary cell, with no passage of dye into neighboring cells. Furthermore, addition of halothane, inhibited connexin activity as represented by disruption of dye transfer between cells (FIG. 4).

Histological Assessment:

Additionally, histological assessment of cardiomyocyte seeded patches between 1, 4 and 8 days after seeding demonstrate that seeded cardiomyocytes adhere to the 3DFC and proceed to align on the surface of the 3DFC. The cardiomyocytes remain on the outer surface of the 3DFC, on the seeded face over time (one to eight days) and do not migrate into the 3DFC or to the opposite, non-seed side. (FIGS. 8-11) Cells centrifuged onto the patch were found to reside between the fibers of the 3DFC.

Echocardiography and Hemodynamics:

Rats were infarcted using standard techniques in our laboratory. In short, a permanent ligature was placed around the left coronary artery of rats and the animals allowed to recover for three weeks. During this recovery time, rats developed chronic heart failure (CHF); classically defined as depressed ejection fraction and cardiac output, elevated end-diastolic pressure and volume with increased left ventricular chamber dimensions. Chronic heart failure rats were divided at random into control (no treatment) and treatment (cardiomyocyte seeded 3DFC) groups 3 weeks after coronary ligation. Both control and treatment groups were studied at baseline, seeded 3DFC was applied 3 weeks post MI, and studied all rats were studied 6 weeks post MI (3 weeks post implant of seeded 3DFC). Rats received 3 and 6 week echocardiography in addition to 6 week hemodynamics. (FIG. 5-7)

Graphical data of these (EF, CI, EDP) show changes/improvements with statistical (p<0.05) analysis between groups as denoted. Data were expressed as mean±standard error (SE). For the physiologic and echocardiographic measurements, the Student t test was used for single comparison of sham versus other study groups. Interactions were tested using two-way analysis of variance (ANOVA), intergroup differences were evaluated using the Student-Newman-Keuls test for statistical significance (P≦0.05).

Using methods previously described, cardiomyocytes were isolated and seeded onto the 3DFC and incubated overnight. Cardiomyocyte seeded 3DFC were then sutured directly onto the epicardial portion of the rat's infarcted left ventricle. The rat's chest was closed and the animal allowed to recover. Echocardiography data demonstrates improvements in ejection fraction (25%) and cardiac output (55%) in treated vs. non-treated rats (FIGS. 5 & 6), thus demonstrating the use of the constructs of the invention for treating CHF. 

We claim:
 1. A method for treating a disorder characterized by a lack of functioning cardiomyocytes, comprising contacting the heart of a subject suffering from such a disorder with an amount effective to treat the disorder of a construct comprising cardiomyocytes, cardiac stem cells, or progenitors thereof adhered to a three dimensional fibroblast construct (3DFC), wherein the construct is capable of spontaneous synchronized contractions across the 3DFC; and wherein the cardiomyocytes, cardiac stem cells, or progenitors thereof are seeded on the construct at a density of between 0.5×10⁶ cells/cm² and 5×10⁶ cells/cm² and/or the cardiomyocytes, cardiac stem cells, or progenitors thereof are present in a ratio of between about 1:10 and about 10:1 with fibroblasts on the 3DFC.
 2. The method of claim 1, wherein the disorder is selected from the group consisting of chronic heart failure (CHF), ischemia without heart failure, cardiomyopathy, dilated cardiomyopathy (DCM), cardiac arrest, congestive heart failure, stable angina, unstable angina, myocardial infarction, coronary artery disease, valvular heart disease, ischemic heart disease, reduced ejection fraction, reduced myocardial perfusion, maladaptive cardiac remodeling, left ventricle remodeling, reduced left ventricle function, left heart failure, right heart failure, backward heart failure, forward heart failure, systolic dysfunction, diastolic dysfunction, systemic vascular resistance, low-output heart failure, high-output heart failure, dyspnea on exertion, dyspnea at rest, orthopnea, tachypnea, paroxysmal nocturnal dyspnea, dizziness, confusion, cool extremities at rest, exercise intolerance, easy fatigueability, peripheral edema, nocturia, ascites, hepatomegaly, pulmonary edema, cyanosis, laterally displaced apex beat, gallop rhythm, heart murmurs, parasternal heave, and pleural effusion.
 3. The method of claim 2, where the disorder comprises CHF.
 4. The method of claim 2, where the disorder comprises DCM.
 5. The method of claim 1, wherein the construct is attached to the epicardium.
 6. The method of claim 5, wherein the construct is non-contracting at the time of contacting with the subject's heart.
 7. The method of claim 1, wherein the contacting comprises contacting the construct to the subject's left ventricle.
 8. The method of claim 1, further comprising contacting the subject's heart with one or more of thymosin beta-4 (TB4), akt murine thyoma viral oncogene homolog (AKT1), stroma cell-derived factor-1 alpha (SDF-1), and hepatocyte growth factor (HGF).
 9. The method of claim 1, wherein the treating comprises one or more of improving left ventricular function, fall in end diastolic pressure (EDP), improving myocardial perfusion, repopulating of the heart's anterior wall with cardiomyocytes, and reversing maladaptive left ventricle remodeling in CHF subjects.
 10. The method of claim 1, wherein the cardiomyocytes or cardiac stem cells are present on the construct in a ratio between about 1:10 and about 10:1 fibroblasts.
 11. The method of claim 1, herein the cardiomyocytes or cardiac stem cells are seeded on the construct at a density of between 1.3×10⁶ cells/cm² and 5×10⁶ cells/cm²
 12. The method of claim 1, wherein the cardiomyocytes or cardiac stem cells are seeded on the construct at a density of between 1.5×10⁶ cells/cm² and 3×10⁶ cells/cm².
 13. The method of claim 1, wherein the cardiomyocytes or cardiac stem cells are of human origin.
 15. The method of claim 1, wherein the cardiomyocytes, cardiac stem cells, or progenitors thereof adhered as a 1-2 cell thick layer over a layer of fibroblasts on (a) the top surface of the 3DFC, wherein the bottom surface of the 3DFC does not have cardiomyocytes, cardiac stem cells, or progenitors thereof adhered to it, (b) the bottom surface of the 3DFC, wherein the top surface of the 3DFC does not have cardiomyocytes, cardiac stem cells, or progenitors thereof adhered to it; or (c) the top and the bottom surface of the 3DFC. 